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Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4 * ♦

View Article: PubMed Central - PubMed

ABSTRACT

Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo. We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level.

No MeSH data available.


Gadd45a TAP, a functional Gadd45a construct designed for tandem affinity purification in mouse skeletal muscle.A, schematic of the Gadd45a TAP construct. B–E, mouse TA muscle fibers were transfected with 20 μg of Gadd45a TAP plasmid plus 2.5 μg of eGFP plasmid. In each mouse, the contralateral TA muscle fibers (Control) were transfected with 20 μg of empty TAP plasmid plus 2.5 μg of eGFP plasmid. Bilateral TA muscles were harvested for analysis 7 days post-transfection. B, skeletal muscle protein extracts were subjected to immunoblot analysis using monoclonal anti-FLAG IgG. C, representative fluorescence microscopy images of muscle cross-sections. D, average diameters of skeletal muscle fibers. Each data point represents the mean of >450 muscle fibers from one muscle, and horizontal bars denote average of the means ± S.E. p value was determined with a paired t test. E, size distribution of all muscle fibers from D.
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Figure 1: Gadd45a TAP, a functional Gadd45a construct designed for tandem affinity purification in mouse skeletal muscle.A, schematic of the Gadd45a TAP construct. B–E, mouse TA muscle fibers were transfected with 20 μg of Gadd45a TAP plasmid plus 2.5 μg of eGFP plasmid. In each mouse, the contralateral TA muscle fibers (Control) were transfected with 20 μg of empty TAP plasmid plus 2.5 μg of eGFP plasmid. Bilateral TA muscles were harvested for analysis 7 days post-transfection. B, skeletal muscle protein extracts were subjected to immunoblot analysis using monoclonal anti-FLAG IgG. C, representative fluorescence microscopy images of muscle cross-sections. D, average diameters of skeletal muscle fibers. Each data point represents the mean of >450 muscle fibers from one muscle, and horizontal bars denote average of the means ± S.E. p value was determined with a paired t test. E, size distribution of all muscle fibers from D.

Mentions: A previously published NMR analysis of Gadd45a revealed a globular protein with a disordered flexible region at its N terminus (19). To develop a Gadd45a construct suitable for tandem affinity purification (TAP),2 we placed two affinity tags (FLAG and S-tag) at the N terminus of Gadd45a, generating a protein that we termed Gadd45a TAP (Fig. 1A). To confirm that the affinity tags in Gadd45a TAP did not interfere with Gadd45a-mediated skeletal muscle atrophy, we transfected a plasmid encoding Gadd45a TAP into the tibialis anterior (TA) skeletal muscles of living mice. In each mouse, the contralateral TA muscle was transfected with empty plasmid and served as an intrasubject control. To detect transfected muscle fibers, bilateral TA muscles were co-transfected with plasmid-encoding eGFP, a transfection marker that does not alter skeletal muscle fiber size. It is important to note that in vivo electroporation transfects differentiated muscle fibers, but not satellite cells or connective tissue cells (20). Seven days after skeletal muscle fiber transfection, we euthanized the mice and harvested bilateral TA muscles for biochemical and histological analyses. As expected, transfection of the Gadd45a TAP plasmid generated an ≈30-kDa Gadd45a TAP protein in mouse skeletal muscle (Fig. 1B). Furthermore, Gadd45a TAP significantly reduced skeletal muscle fiber size (Fig. 1, C–E), indicating that Gadd45a TAP induces skeletal muscle fiber atrophy in vivo, similar to untagged Gadd45a.


Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4 * ♦
Gadd45a TAP, a functional Gadd45a construct designed for tandem affinity purification in mouse skeletal muscle.A, schematic of the Gadd45a TAP construct. B–E, mouse TA muscle fibers were transfected with 20 μg of Gadd45a TAP plasmid plus 2.5 μg of eGFP plasmid. In each mouse, the contralateral TA muscle fibers (Control) were transfected with 20 μg of empty TAP plasmid plus 2.5 μg of eGFP plasmid. Bilateral TA muscles were harvested for analysis 7 days post-transfection. B, skeletal muscle protein extracts were subjected to immunoblot analysis using monoclonal anti-FLAG IgG. C, representative fluorescence microscopy images of muscle cross-sections. D, average diameters of skeletal muscle fibers. Each data point represents the mean of >450 muscle fibers from one muscle, and horizontal bars denote average of the means ± S.E. p value was determined with a paired t test. E, size distribution of all muscle fibers from D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5016147&req=5

Figure 1: Gadd45a TAP, a functional Gadd45a construct designed for tandem affinity purification in mouse skeletal muscle.A, schematic of the Gadd45a TAP construct. B–E, mouse TA muscle fibers were transfected with 20 μg of Gadd45a TAP plasmid plus 2.5 μg of eGFP plasmid. In each mouse, the contralateral TA muscle fibers (Control) were transfected with 20 μg of empty TAP plasmid plus 2.5 μg of eGFP plasmid. Bilateral TA muscles were harvested for analysis 7 days post-transfection. B, skeletal muscle protein extracts were subjected to immunoblot analysis using monoclonal anti-FLAG IgG. C, representative fluorescence microscopy images of muscle cross-sections. D, average diameters of skeletal muscle fibers. Each data point represents the mean of >450 muscle fibers from one muscle, and horizontal bars denote average of the means ± S.E. p value was determined with a paired t test. E, size distribution of all muscle fibers from D.
Mentions: A previously published NMR analysis of Gadd45a revealed a globular protein with a disordered flexible region at its N terminus (19). To develop a Gadd45a construct suitable for tandem affinity purification (TAP),2 we placed two affinity tags (FLAG and S-tag) at the N terminus of Gadd45a, generating a protein that we termed Gadd45a TAP (Fig. 1A). To confirm that the affinity tags in Gadd45a TAP did not interfere with Gadd45a-mediated skeletal muscle atrophy, we transfected a plasmid encoding Gadd45a TAP into the tibialis anterior (TA) skeletal muscles of living mice. In each mouse, the contralateral TA muscle was transfected with empty plasmid and served as an intrasubject control. To detect transfected muscle fibers, bilateral TA muscles were co-transfected with plasmid-encoding eGFP, a transfection marker that does not alter skeletal muscle fiber size. It is important to note that in vivo electroporation transfects differentiated muscle fibers, but not satellite cells or connective tissue cells (20). Seven days after skeletal muscle fiber transfection, we euthanized the mice and harvested bilateral TA muscles for biochemical and histological analyses. As expected, transfection of the Gadd45a TAP plasmid generated an ≈30-kDa Gadd45a TAP protein in mouse skeletal muscle (Fig. 1B). Furthermore, Gadd45a TAP significantly reduced skeletal muscle fiber size (Fig. 1, C–E), indicating that Gadd45a TAP induces skeletal muscle fiber atrophy in vivo, similar to untagged Gadd45a.

View Article: PubMed Central - PubMed

ABSTRACT

Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo. We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level.

No MeSH data available.